Method of tissue repair

ABSTRACT

A method for joining tissue comprising aligning and abutting edges of the tissue to be joined applying a biodegradable, biological solder or an analogue thereof, across the edges and exposing the solder to an energy source under conditions which provide transfer of energy from the source to the solder to cause the solder to bond to the tissue surface adjacent the edges to provide a weld holding the edges together.

TECHNICAL FIELD

[0001] The present invention relates to methods for joining livingtissues, including veins, arteries, microvessels, tubes, nerves, organtissues and biological surfaces, such as peritoneum, omentum, fascia,shin, artificial tissues, and to pharmaceutical products useful injoining these tissues.

BACKGROUND ART

[0002] Joining tissues such as veins, arteries, microvessels, tubes,nerves, tissues and biological surfaces such as the peritoneum and skinhas mainly been carried out clinically to date by suturing andmicrosuturing.

[0003] Microsuturing requires considerable skill and is a time consumingprocedure. Frequently, tissues which have been joined by microsuturingform considerable scar tissue. Some of the difficulties encountered withmicrosuturing can be better understood by considering the example ofrejoining damaged peripheral nerve tissue.

[0004] Peripheral Nerves

[0005] The electrical signals that control the body's organs andtransmit information back and forth to the central nervous system (CNS)travel along peripheral nerves. The structure of these peripheral nervesis analogous to telephone cables. In a telephone cable there is a strongprotective outer coating that protects all the inner components. Thecopper wires are often grouped in separate insulating tubes that lead todifferent systems. Each of the inner copper wires is a single line thatcan transmit electricity in either direction and has an insulatingcoating around it so that it does not interfere with the lines next toit.

[0006] A peripheral nerve (FIG. 1) has an outer membrane consisting ofconnective tissue such as collagen. This membrane (epineurium) protectsand holds the separate nerve bundles together. The nerve bundles whichlie inside this membrane are called fascicles. These fascicles also havea collagen based surrounding membrane and their task is to grouptogether nerve axons supplying a similar area of the body. Inside thefascicle membrane the axons are surrounded by loose connective tissue.The axons are a long extension from a cell body which is containedwithin the CNS in the spine or the brain. Sensory axons transmit to theCNS and motor axons transmit from the CNS. Nerve metabolism is sustainedby the vascular system from both outside the nerve and along the centreof the nerve.

[0007] Peripheral nerves can have very small diameters. For instance,the mature median nerve at the wrist is approximately 1 cm in diameterand contains an average of forty fascicles, each of which can contain upto 4500 axons. When a peripheral nerve is cut all axons distal to thewound change their properties as axon flow is cut off from the cellbody. Even when the nerve is reconnected, these axons continue todegenerate distally. The Schwann cells which normally wrap themselvesaround the axons as insulation guide regenerating axons. Joining nervesas accurately as possible by lining up corresponding fascicles enablesthe axons to more efficiently regenerate.

[0008] Operating upon nerves has been facilitated by using magnificationand special microsurgical equipment. Accurate repairs need to beeffected at the fascicular level ensuring that regeneration is along thecorrect bundle leading to the original area those axons supplied. Thecurrent technique of peripheral nerve repair uses microsuturing (FIG.2). This technique requires a dedicated, trained surgeon asmicrosuturing of just one of the many fascicles with three or moremicrosutures (using say a 70 micron diameter needle and 30 micronthread) can take very long operating times.

[0009] Microsuturing is at present clinically used where the skills areavailable. Unfortunately, there are relatively few surgeons who have thenecessary manipulative skills for operating at high magnification. Evena reasonable microsuturing technique results in long operating timeswith added damage to the inner axons due to sutures penetrating the thininsulating perineurial sheath. The use of sutures results in somescarring of the repair due to foreign body reaction. There is alsoevidence which indicates that in the long term scar tissue formation andscar maturation can lead to impairment of the joined nerve.

[0010] Work has been performed on the use of lasers alone in effectingnerve joins. One of the problems of laser welding has been the fact thatthe intact gel-like nerve tissue of the axons is actually under pressurewithin the fascicle. When the fascicle is cut this material extrudes.This can lead to the direct laser weld being formed on nerve tissuerather than the surrounding membrane of the fascicle, causing nervedamage. To date the welds have typically been made using infrared laserssuch as CO₂ lasers which rely on water absorption for energy transfer.Tissue preparation before welding relies on overlapping the nervemembranes. This is difficult due to the extruding gel-like axons and socan lead to denaturation of the nerve axon material. The affected tissuetends to scar and the fibrous tissue that proliferates as a result is apoorer electrical conductor than nerve tissue. The bonds formed to dateas described in the prior art using laser welding have typically lackedstrength. These laser joins alone tend to fail so microsuturing has beenused in addition to welding to strengthen these joins.

[0011] To deal with at least some of the deficiencies of laser welding,various glues have been used in forming the welds. These low proteinconcentration, fluid glues tend to run between the ends of the nervethat are being joined which may result in damage to the axoplasm of thenerve fascicle and also hinder regeneration. They are also appliedaround the join which is then circumferentially welded. These joinslater show thick scarring which causes stricture of the nerve. Moreover,the joins tend to be weak.

[0012] The welding techniques so far available also tend to lackprecision. Factors that influence the precision of this approachadversely include differences in: the consistency of the glue used; theaperture of the needle or other device used to apply the glue; and thepressure exerted in applying the glue.

DESCRIPTION OF THE INVENTION

[0013] The present invention provides a method for joining tissuecomprising:

[0014] aligning and abutting edges of the tissue to be joined;

[0015] applying a solder, across the aligned and abutted edges; and

[0016] exposing the solder to an energy source under conditions whichprovide a transfer of energy from the source to the solder to cause thesolder to bond to the tissue surface adjacent the edges thus providing aweld holding the edges together.

[0017] In addition to causing the solder to bond to the protein of theunderlying tissue, the energy transfer can affect the structure of thesolder itself leading to bonding within the solder and an enhancement ofthe strength of the solder and hence the join.

[0018] Drops of solder are typically used where the solder is a fluidsolder, and are “painted” across the edges.

[0019] The solder can also be provided as a preformed solid strip.

[0020] The energy source is typically a laser.

[0021] A variety of tissue types can be joined using this method. Themethod is applicable to anastomoses of biological tubes including veins,arteries, lymphatics, nerves, vasa efferentia, fallopian tubes, bileducts, tubes of the alimentary canal, the ureter, the urethra, tearducts, bronchi and any other such bodily tubes as well as to repairs ofincisions or tears of biological organs such as kidneys, liver orspleen, or of biological surfaces such as the peritoneum and skin. Itwill therefore be understood that the method can be used in a variety ofjoin situations including the joining of cylindrical anastomoses and theclosure of linear defects such as incisions.

[0022] Where the tissue repair is with respect to nerve tissue or othertissue tubes where the tube contents need to be protected from damage,it is especially important that the weld should not be concentrated onthe edges being joined as this can damage extruded tissue. Rather, theweld should be distributed across the planar or tubular surface in whichthe discontinuity lies.

[0023] Where the tissue to be repaired is an essentially hollow bodytube such as a blood vessel, the repair can additionally comprise theinsertion of a thin-walled hollow cylinder of solder inside the tubeunder repair so that the cylinder spans the severed portions of thetube. Typically, while the severed tube and cylinder assembly is heldtogether, energy from the energy source is directed through the tubewall to bond the cylinder to the tube ends. The cylinder may incorporatea dye, as hereinafter described, to attract energy to the cylinder formore efficient welding. The repair is completed by the application of atleast one strip or drop of solder across the edges on the outer surfaceand treating the applied solder as described above.

[0024] Where the repair is with respect to tissue surfaces such asperitoneum, it will be understood that it is less important to avoidconcentration of welding on the edges.

[0025] The method can also be modified for the repair of otherdiscontinuities in tissue surfaces such as holes, resulting fromaccident or surgery. In this form of the invention the solder may bespread or pre-cut to conform to the shape of the repair site, and theedges of the repair site may not need to be aligned or abutted for therepair to be effected.

[0026] A typical nerve repair using the method of the invention is onein which the edges are ends of a cut peripheral nerve fascicle that areto be joined together or an end of a nerve fascicle and the fascicle ofsubstitute nerve graft material. This latter situation is particularlyapplicable where nerve repair is required but a section of the nerveunder repair has been severely damaged or is unavailable, so that theavailable ends of the fascicle are too remote from each other to bedirectly joined. The actual nature of the damage sustained by the nerveand whether the repair is a primary or secondary repair are factorsaffecting recovery but in any case the edges of nerve fascicles to bejoined are cleanly cut at right angles prior to joining.

[0027] Application of the solder as a strip or strips, with spacebetween for natural co-aptation of the surfaces themselves permits thenerve under repair to revascularise. Circumferential welding, bycomparison, can inhibit the body's natural healing process and so slowdown blood capillary access needed for the area of repair. Lasersoldering and suturing techniques ultimately rely on the bodyregenerating connective tissue to hold the nerve together after eithersolder or suture connections break down and are replaced by the healingprocess. The present inventors have shown in in vivo experiments thatsuccessful regeneration can be achieved by the methods of the presentinvention without restriction on surrounding tissue movement after theoperation. In the case of nerve repair operation on human patients it isroutine to initially restrict the movements of the joints of theoperated limbs to assist in reducing tension across the repair site.

[0028] Typical biodegradable, biological solders useful in the method ofthe invention include protein solders.

[0029] It is envisaged that other naturally occurring biomolecules couldbe used as alternatives. Further analogues of biological, biodegradablepolypeptides could be used. Analogues of biological, biodegradablepolypeptides useful in the invention include synthetic polypeptides andother molecules capable of forming a viscous “glue” that does not reactadversely within the tissue undergoing repair.

[0030] The protein solder may be a solid or a fluid solder composition.

[0031] Fluid protein solder compositions useful in strip weldingtypically comprise between 100 and 120 mass % of protein relative towater. Preferably, fluid protein solders comprise between 100 and 110mass % protein relative to water.

[0032] The fluid solder strip is typically 50 to 200 μm in thickness.Its length is selected to suit the join to be formed but typically is ofthe order of 2 to 3 mm in length. It is typically painted across thejoin.

[0033] Solid protein solder compositions useful in strip weldingtypically comprise between 120 and 230 mass % protein relative to water.Preferably the strip comprises 170 to 230 mass % protein and morepreferably about 210 mass %.

[0034] It will be understood that different proteins will have differentdegrees of solubility in water or appropriate solutions which in turnwill affect the optimum concentration of protein in the composition fordifferent protein solders. Appropriate ranges for particular proteins inboth solid and fluid solders can be determined based on the knownproperties of the proteins.

[0035] Typically, the solid protein solder composition is provided as apreformed strip. Solid solder strips are easier to manipulate than fluidsolders. Under the moist conditions inherent in surgery fluid soldersmay run making it difficult to laser denature the solder before it hasspread. The solid solder strips can have a paste like or more rigidconsistency. They are typically placed across the join withmicroforceps. In one form of the invention, it is envisaged that thesolder strips will be substantially rectangular in shape. However,different shape strips may be required in different repair situations.It may also be desirable to provide a plurality of strips joinedtogether for efficient repair of a large or a substantial number ofrepair sites.

[0036] The protein solder may comprise a single protein of which albuminis a typical example or alternatively the solder may comprise more thanone protein.

[0037] Albumin has desirable qualities for solid solder strip formationsince it has a high proportion of β sheet structure which gives rigidityto the strips. Fibrin is another example of a protein with significant βsheet structure. Incorporation of α helical protein in the solder canassist in making the strips more malleable and thus retain a flatterprofile which is particularly well suited for joining nerve ends. Anexample of a suitable proportion of α helical protein is between 1 and10% by weight of the protein used. About 5% is a preferred amount.Collagen, tropoelastin and elastin are examples of suitable a helicalproteins.

[0038] Protein used in the solder is selected to minimise the risk ofadverse host reactions and should therefore preferably be an autologousprotein for the host or a foreign protein of low antigenicity.

[0039] The proteins may be obtained from any suitable source.Recombinantly or synthetically produced proteins as well as purifiednaturally occurring proteins may be used.

[0040] Preferably, when the solder is to be used with a laser whichproduces energy at a suitable wavelength the composition includes asubstance, such as a dye, which absorbs energy at the wavelengthproduced by the laser with which the solder is to be used. It ispreferable to choose the combination such that the dye or othersubstance absorbs the energy transmitted by the laser efficiently butthe underlying tissue to be joined absorbs the transmitted energypoorly. The dye or other substance assists in making the weldingspecific to the solder used which in turn assists in minimisingaccidental tissue heating damage to the underlying tissue.

[0041] The process of bonding, where protein solders are used, relies onprotein molecules being available for cross-linking. This occurs whenthe protein molecules are unfolded. Upon laser irradiation of, forinstance, an albumin and indocyanine green containing solder at a nervetissue join, albumin molecules are heated through energy transfer fromthe indocyanine green molecules, allowing them to unfold and bondbetween themselves and to neighbouring tissue surface such as thefascicle membrane.

[0042] Dyes which contrast with the tissues being repaired can also beuseful in making the solder easier to see. An example of a dye with thisproperty is indocyanine green.

[0043] When the laser used is a CO₂ laser, a dye will not assist theenergy transfer, as the energy transfer is by water absorption.

[0044] The energy provided by the energy source should be sufficient tobond the solder to form the weld while minimising damage to theunderlying tissue. The temperature required to denature a protein solderis typically at least 50° C. and may exceed 100° C. A preferred range is50° to 90° C. A particularly preferred range is 80° to 90° C.

[0045] The time of treatment for each join to be effected can varydepending on such factors as ambient conditions, altitude, and of coursethe nature of the tissue to be joined. The duration of treatment istypically short. A 30 second passage for laser treatment of a 0.4 mgstrip is an example of the time involved although it will be understoodthat shorter or longer treatment times could be required. It will beunderstood that solid solder takes longer to denature than fluid solder.

[0046] In a second aspect the present invention provides a proteinsolder composition comprising protein and a suitable solvent for theprotein. Water is typically used as the solvent for water solubleproteins.

[0047] In a third aspect the present invention provides a kit for use injoining tissues comprising, in a preferably sterile pack, a plurality ofprotein solder strips and/or shapes of the second aspect of theinvention. Preferably a plurality of strip lengths and/or shape sizesare included in the pack.

[0048] The kit preferably includes means for sterile manipulation of thestrips. The kit also preferably includes means for measuring the strips.

[0049] The kit may also comprise an energy source such as a fibrecoupled laser system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1 shows the structure of a peripheral nerve in schematicform.

[0051]FIG. 2 shows the joining of a peripheral nerve by prior artmicrosuturing techniques.

[0052]FIGS. 3a) and b) shows in schematic form joining of a nervefascicle with a) fluid solder and b) solid strips.

[0053]FIG. 4 shows the repair site of a 0.3 mm diameter tibial nerveimmediately after: a) diode laser strip welding, and b) microsuturing.

[0054]FIG. 5a shows a rat tibial nerve welded by the laser soldermethods of the present invention. The solder and the membrane aredenatured but no significant change to the axons has occurred (x100Giemsa).

[0055]FIG. 5b shows a rat sciatic nerve joined by microsuturing using10-0 nylon. Localised perineural and axonal damage occurs.

[0056]FIG. 6 shows in schematic form joining of a blood vessel usinginternal biodegradable solid solder cylinder and external solid solderstrips.

[0057]FIG. 7 shows in schematic form a cross-section of a repaired nervefascicle.

[0058]FIG. 8 shows the method used for measuring tensile strength ofrepaired nerves.

[0059]FIG. 9 shows a solid solder strip positioned upon a severed rattibial nerve just prior to laser welding.

[0060]FIG. 10a shows regeneration of myelinated axons in a laser nerverepair that has regenerated for 3 months.

[0061]FIG. 10b shows fibrous tissue around a suture in a sutured nervethat has regenerated for 3 months.

[0062]FIG. 11 shows muscle action potential results for repaired nerves.

BEST METHOD OF CARRYING OUT THE INVENTION

[0063] Tissue repair is performed using a laser to activate a proteinsolder applied across the tissue edges to be joined. This solderdenatures upon laser irradiation and bonds with itself and theneighbouring membrane to form the join. The procedure is shownschematically in FIGS. 3 and 7 for a repair to a nerve fascicle. Thesolder is applied in longitudinal strips across the join.

[0064] Nerve Repair

[0065] Repair to severed nerve tissues is effected by the placement ofsolder across the severed edges and exposure of the solder to laser asdescribed above. In order to repair nerve tissue without damage to thecontents of the nerve it is desirable to avoid concentrating the weld onthe edges as extruded nerve contents may be damaged. Rather the weldshould be distributed across the planar or tubular surface in which thediscontinuity lies.

[0066] Hollow Body Tube Repair

[0067] When repairing hollow body tubes it is preferable to insert aninternal cylinder of solder into the tube so that it lies between thediscontinuity. The severed ends of the tube are placed over oppositeends of the solder cylinder. The arrangement is shown in FIG. 6.Lasering can then be effected to cause bonding of the cylinder to thetube being joined while the arrangement is held in place. If there is agood fit between the tube and the cylinder this laser step may not berequired. The join is completed by the addition of external solder asfor nerve repairs.

[0068] Tissue Surface Repairs

[0069] Surfaces such as peritoneum are planes of tissue in which joinswithout sutures can be achieved by the application of solder across thediscontinuities to be joined and welding as described above. In thiscase it is less important to avoid concentration of welding on theedges.

[0070] Laser and Solder System Suited to Nerve Fasicular Repair

[0071] To denature the protein solder, a GaAs/GaAlAs laser diode with anominal power of 250 mW (Spectra Diode Labs, San Jose, Calif.) is used.The laser light is coupled into a 100 μm diameter core optical fibrewhich is hand held in a fibre chuck. The diode is operated in continuousmode at 75 mW during the laser soldering. Because this laser is Class3b, and is not eye safe, protective glasses must be worn at all timeswhen using this laser.

[0072] A suitable protein solder is a mixture of water, albumin andindocyanine green (ICG) dye (Becton Dickinson, Mo.). Indocyanine greenhas a maximum absorption coefficient at a wavelength of 805 nm of 2×10⁵M⁻¹ cm⁻¹. The percentages of albumin and dye compared to the water were110% and 0.6% respectively for fluid solder. 210% albumen was used inpreparing solder strips. It is notable that ICG dye appears topreferentially bind with the albumin ensuring that heat is efficientlytransferred to denature the protein solder.

[0073] Laser Soldering Technique

[0074] When conducting the surgery an operating microscope or some formof magnification is preferable. For a laser solder repair of a tubularjoin a section of thin gauze material is placed under the join to assistin a rotation technique. The tissue edges are prepared in accordancewith standard techniques for the tissue type and geometry of the repair.

[0075] Using micro forceps the edges are aligned and butted together. A2 mm long stripe of fluid solder is “painted” longitudinally across thejunction of the edges using a 30 gauge needle freshly coated in thesolder. Alternatively a strip solder is laid across the join usingmicroforceps. The solid strip repair method is simpler. A solid strip isheld in special microforceps and placed across the junction parallel tothe length of the structures to be joined. The laser output is thendirected at the solid strip and the solid solder changes coloursignalling denaturation which causes it to adhere to the underlyingtissue membrane. The process is repeated with further strips to ensure astrong union of surface.

[0076] The diode laser output from the 100 μm optical fibre is then usedin a 30 second continuous pass to denature solid solder into a stripweld. At a diode output power of 75 mW, the solid solder strip turnsbrown on the surface and opaque underneath from the single pass,signalling denaturation. When using fluid solder denaturation occursmore quickly. A two second laser pass can be sufficient to denature thefluid solder. Generally a second layer of fluid solder is applied to thestrip in order to increase the strength of the weld and the two secondlaser pass is repeated. The gauze under the join is then used with themicro-forceps to rotate the join so that other strips can be applied.

[0077] Preparation of Fluid Protein Solder

[0078] Composition

[0079] Albumin (fraction V powder from Sigma, St. Louis, Mo.) at least100% to 110% by weight compared to water.

[0080] Indocyanine Green (Becton Dickinson, Mo.) approximately 0.6% byweight compared to water.

[0081] Water (injection grade)

[0082] Procedure

[0083] A solution of ICG in water was prepared in a minitube. Thealbumin was added to the tube. The albumin and solution were mixed usinga vortex mixer. This causes the protein structure to change leading tolinkage of protein molecules to each other rather than to watermolecules.

[0084] Preparation of Solid Protein Solder

[0085] Composition

[0086] Albumin (fraction V powder from Sigma, St. Louis, Mo.) 210% byweight compared to water

[0087] Indocyanine Green (Becton Dickinson, Mo.) approximately 0.6% byweight compared to water

[0088] Water (injection grade)

[0089] Procedure

[0090] The ICG was dissolved in the water and the albumin was added tothis solution in a minitube. This combination was mixed using a vortexmixer and a needle. The combination was mixed (for approximately 3minutes) until it became a homogenous, malleable, green paste. The phaseof the mixture changed under this mixing technique to provide an almostsolid composition with mainly protein to protein linkages rather thanprotein to water linkages. The system is no longer a solution at thisstage. The protein paste was malleable and could be cut into strips forup to about 30 minutes after mixing. After this time the paste hardeneddue to dehydration and became too hard to cut.

[0091] The resulting strips were between 50 and 100 μm in thickness,about 0.6 mm wide and 1.5 to 3.5 mm long. It will be understood thatwhere the strips are used in mending nerve fascicles that the desiredwidth and length are dictated by fascicle dimensions. The width,thickness and length mentioned here are those suitable for use with arat tibial nerve which has a diameter of 0.2 to 0.8 mm. The ratio ofstrip width to nerve circumference is typically:

[0092] Width ^(˜)1/5 circumference

EXAMPLE 1

[0093] A 100 μm core optical fibre-coupled 75 mW diode laser operatingat a wavelength of 800 nm has been used in conjunction with a proteinsolder to stripe weld severed rat tibial nerves, reducing the longoperating time required for microsurgical nerve repair. Welding isproduced by selective laser denaturation of the protein based solderwhich contains the dye indocyanine green. Operating time for lasersoldering was 10+/−5 min. (n=24) compared to 23+/−9 min. (n=13) formicrosuturing. The laser solder technique resulted in patent welds witha tensile strength of 15+/−5 g, while microsutured nerves had a tensilestrength of 40+/−10 g. Histopathology of the laser soldered nerves,conducted immediately after surgery, displayed solder adhesion to theouter membrane with minimal damage to the inner axons of the nerves. Anin vivo study, with a total of fifty-seven adult male wistar rats,compared laser solder repaired tibial nerves to conventional microsuturerepair. Twenty-four laser soldered nerves and thirteen sutured nerveswere characterised at three months and showed successful regenerationwith average Compound Muscle Action Potentials (CMAP) of 2.4+/−0.7 mVand 2.7+/−0.8 mV respectively. Histopathology of the in vivo study,confirmed the comparable regeneration of axons in laser and sutureoperated nerves. A faster, less damaging and long lasting laser basedanastomotic technique is presented.

Materials and Methods

[0094] 1. Animals

[0095] A total of fifty-seven young adult male Wistar rats weighingbetween 400 and 550 g at the outset were used in this study. Thirty-fourrats received laser solder repair and the remaining twenty-threereceived standard microsuture repair as detailed below. Five rats ofeach repair method were used for tensile strength measurements and lightmicroscopy immediately after surgery and the remaining thirty-seven ratswere subjected to a study of functional recovery using electrophysiologyand histopathology.

[0096] 2. Laser Solder System

[0097] To denature the protein solder, a GaAs/GaAlAs laser diode with anominal power of 250 mW (Spectra Diode Labs, San Jose, Calif.) was used.The laser light was coupled into a 100 μm diameter core optical fibrewhich was hand held in a fibre chuck. The diode laser was mounted on aheat sink, and the diode current and temperature were controlled by aSDL-800 diode driver. The diode was operated in continuous mode at 75 mWduring the laser soldering, corresponding to a maximum power density of955 W/cm at the tissue. The laser output power was measured with aScientech (Boulder, Colo.) power meter. Because this laser is Class 3b,and is not eye safe, protective glasses were worn at all times whenusing this laser.

[0098] The solder used in this study was an albumin based proteinmixture, also containing indocyanine green (ICG) dye (Becton Dickinson,Mo.). Indocyanine green has a maximum absorption coefficient at awavelength of 805 nm of 2×10⁵ M⁻¹ cm⁻¹. It is notable that this dyeappears to preferentially bind with the proteins ensuring that heat isefficiently transferred to denature the protein solder.

[0099] 3. Surgery

[0100] Anaesthesia was maintained during surgery using a mixturecontaining Fluothane (4% during induction, 2% thereafter) in O₂ (1L/min). Using a OPMI 7 operating microscope (Zeiss, West Germany) thesciatic nerve of the left leg was exposed at the sciatic notch so thatthe nerve branches could be distinguished. The tibial branch, just belowthe sciatic notch, was exposed from the surrounding subcutaneous tissuefor a length of 1 cm. For a laser solder repair, a section of thin gauzematerial was placed under the tibial nerve to assist in rotation of thenerve, and for the suture repair, a section of plastic was placed underthe nerve to allow easier suturing. The tibial nerve was then severedwith serrated micro-scissors and left for 3 minutes for the normalextrusion of axoplasm to occur. This was then trimmed with the serratedmicro-scissors as required, after which the nerve was repaired witheither four laser solder strips or four 10-0 perineurial sutures.

[0101] The laser solder method involved aligning both stumps of thesevered nerve with micro-forceps then a 2 mm long strip of solder was“painted” longitudinally across the junction of the severed ends using a30 gauge needle freshly coated in the solder (FIG. 3a). The diode laseroutput from the 100 μm optical fibre was then used in a continuous twosecond pass to denature the solder into a strip weld. At a diode outputpower of 75 mW, the solder was observed to turn brown on the surface andopaque underneath from the single pass, signalling denaturation. Asecond layer of solder was applied to the strip and the two second laserpass was repeated. The gauze under the nerve was then used with themicro-forceps to rotate the nerve so that three other two layeredstripes could be applied, each approximately 90° apart.

[0102] Seven rats were operated with a more advanced version of theorganic solder, which is still an albumin based protein mixture but ithas the advantage to be dehydratated and cut into solid rectangularstrips (FIG. 9). The average surface area of the solder strips was1.5+/−0.5 mm² and the thickness was 0.15+/−0.01 mm. Four strips werepositioned along the tibial anastomized nerve and then radiated with thesame procedure adopted for the fluid solder. The solid strip was fusedwith the perineurium of the tibial nerve by the laser radiation, joiningthe extremites of the sectioned nerve.

[0103] For all operation the time of anastomosis was recorded and aphotographic record was taken for later reference. The animals wereplaced in their cages with no restriction of movement for 3 months.

[0104] 4. Immediate Measurement of Tensile Strength and Histopathology

[0105] In ten of the operated rats, the 1 cm long section of the laserand suture repaired nerves was harvested immediately for tensilestrength measurements. Fine silk was tied to each end of the tibialnerve. One end was then attached to a calibrated force transducer(FT30C, Grass Instruments, Quincy, Mass.) and the other to a screwdriven translator (FIG. 8) As the screw was turned the translator wouldstretch the nerve in a slow and steady manner. The applied tension wasobserved on an oscilloscope connected to the output of the forcetransducer. Tension was applied until the nerve separated, and thebreaking force was recorded. The nerves were kept moist, as upon drying,the tensile strength can be increased.

[0106] For light microscopy the anastomosis site of the tibial nerveswere fixed in 5% formalin, alcohol dehydrated, imbedded in paraffin,longitudinally sectioned and stained with either Masson's trichrome orGiemsa.

[0107] 5. Functional Assessment: Histopathology and Electrophysiology

[0108] Three months post operatively the rats were reanaesthetised usingthe method described in section 3. The site was exposed and theanastomosis of the tibial nerve observed. The two other branches of thesciatic nerve, the peroneal and sural nerves were then severed so thatonly the tibial nerve branch of the sciatic nerve could conductelectrical stimulation of the sciatic nerve to the muscles of the hindfoot. Two days later the rats were positioned on their side andinsulated from the table by a folded surgical drape. An infrared lampwas used to maintain their rectal temperature above 36° C.

[0109] A clinical electromyograph (Cadwell Sierra EMG/EP). was used forstimulation and recording. Two 25 gauge stimulating electrodes wereplaced 10 mm apart on each side of the sciatic nerve above the sciaticnotch, near the hip. The nerve was activated using rectangular pulses(0.1 to 0.3 ms; 0 to 30 mA; 1 Hz). Compound muscle action potentials(CMAPs) were recorded from the plantar muscles of the foot in responseto supramaximal stimulation of the sciatic nerve. A set of threerecording electrodes were used. A 25 gauge ground electrode was insertedsubcutaneously between the stimulating and recording electrodes ^(1,2).A 30 gauge reference electrode was inserted into the heel pad and a 30gauge recording electrode was inserted into the plantar muscles of thefoot . The CMAPs were recorded and processed to determine their negativewave peak value.

[0110] Histopathology of the sutured and laser soldered nerves, wasconducted after the Electrophysiology test with the same procedure asadopted in section 4.

Results

[0111] At the completion of surgery all anastomoses were successful. Theoperating procedure was found to be easier for laser soldering than formicrosuturing. This resulted in the shorter operating times for lasersolder repairs {10±5 min (n=24)} than {23±9 min (n=13)} for microsuturerepairs. The tensile strength of five laser solder repaired nervesimmediately after the operation was 15±5 g and the tensile strength ofthe microsutured nerves, 40±10 g.

[0112] Histopathological examination of the anastomosis sitesimmediately after surgery demonstrated that the albumin and ICG dyebased laser solder does bond well with the outer membrane of the nerve,the perineurium, while the inner axons remain unheated. In FIG. 10a, atibial nerve fascicle weld produced by the diode laser and albumin/ICGdye solder is shown in section. Both the protein solder and theperineurium have denatured forming the bond. On the lower side of thebond, the axoplasm has its normal wavy structure. Note that sinceheating is concentrated at the dye, only denaturation of the solder andadjacent perineurium occurs.

[0113] One of the promising aspects of laser anastomosis is thepotential for reduced damage to the axoplasm by removing the need forsutures. A section showing the effect of microsuturing nerve fasciclesusing 10/0-nylon is shown in FIG. 5b. This section stained with Giemsa,displays axon extrusion at the join, as well as localised perineurialand axonal damage due to the suture.

[0114] Histopathology at 3 months shows regeneration of myelinated axonsin laser nerve repairs (FIG. 10a), with no discontinuity of either thefibers and their sheaths, or the fibrous perineurium. No evidence isseen of inflammation or myelin phagocytosis. Full restoration, asassessed by light microscopy, of the histologic integrity of the tibialnerve has been achieved by the laser weld.

[0115] The sutured nerves also show successfull anastomosis withmyelinated axon regeneration, however, it is still evident that thenylon thread is surrounded by fibrous tissue, which creates an obstacleto the directionality of the regenerated axons (FIG. 10b).

[0116] The electrophysiological measurements of the in vivo study wereperformed on twenty-four laser solder repaired rats and thirteenmicrosuture repaired rats having three months recovery. Of this groupall twenty-four laser solder anastomoses were patent as were thethirteen microsuture anastomoses. The average amplitude of the muscleaction potentials, resulting from supramaximal stimulation of the nerveabove the repair site was 2.4+/−0.7 mV for the twenty-four lasersoldered tibial nerves and 2.7+/−0.8 mV for the thirteen microsuturednerves. The normal muscle action potential produced by stimulating thetibial nerve supramaximally was recorded at 8.7±3 mV from ten rats (FIG.11).

Discussion

[0117] Clinically, when a major peripheral nerve is severed, forty ormore fascicles may need to be individually rejoined. With three or fourmicrosutures per fascicle, suturing tends to be prolonged, as it must bemeticulous. In a nerve graft, where two anastomoses are needed, thesuturing time is doubled. We have sought a suitable method of nerveanastomosis that could at least duplicate the end result but wassignificantly faster than the present hand sewing microsuture technique.A bonus of the described laser soldering method was the demonstratedlack of change to the axonal components beneath the denaturedperineurial layer seen immediately after surgery. Three months latercomparable regeneration was demonstrated by electrophysiological nerveconduction studies.

Industrial Applicability

[0118] The present invention has application in the field of surgerywhere it is of application in joining together tissue edges, in end toend, side to end and side to side applications.

References

[0119] 1) R. Malik, S. Ho and D. B. Church: A new method for recordingand analysing evoked motor potential from dogs. Journal of Small AnimalPractice (1989) 30, 13-19.

[0120] 2) R. Malik, S. Ho: Motor nerve conduction parameters in the cat.Journal of Small Animal Practice (1989) 30, 396-400.

[0121] 3) Laser activated protein bands for peripheral nerve repair. ALauto, R Trickett, R Malik, J Dawes, E Owen. European Biomedical OpticsWeek—BIOS Europe 195 12-16 September 1995 (Proceeding in Press)

1. A method for joining tissue comprising: aligning and abutting edgesof the tissue to be joined; applying a biodegradable, biological solderor an analogue thereof, as herein defined, across the edges; andexposing the solder to an energy source under conditions which providetransfer of energy from the source to the solder to cause the solder tobond to the tissue surface adjacent the edges to provide a weld holdingthe edges together.
 2. The method of claim 1 wherein the tissue is nervetissue and the edges are ends of a peripheral nerve fascicle or a nervefascicle and nerve graft material, and welding is-not effected along theline of discontinuity so as to protect nerve tissue from damage.
 3. Themethod of claim 1 wherein the tissue is an anastomosis of a biologicaltube including veins, arteries, lymphatic, vasa efferentia, fallopiantubes, bile ducts, tubes of the alimentary canal, the ureter, theurethra, tear ducts or bronchi, and wherein a hollow cylinder of thesolder is inserted into the tube between the discontinuous ends prior tothe application of solder to the external surfaces of the tube beingjoined.
 4. A method according to claim 3 wherein the discontinuous endsare held in place while energy is applied to the cylinder within thetube to cause the cylinder to bond with the inner surface of the tube.5. The method of claim 1 wherein the tissue is a repair of an incisionor tear of a biological organ including kidneys, liver or spleen, or ofa biological surface such as the peritoneum or skin.
 6. A method forrepairing a discontinuity in a tissue surface comprising: applying abiodegradable, biological solder or an analogue thereof as hereindefined to the discontinuity; and exposing the solder to an energysource under conditions which provide transfer of energy from the sourceto the solder to cause the solder to bond within itself and to thetissue surface to provide a weld holding the solder and tissuessurrounding the discontinuity together.
 7. The method of claim 1 or 2wherein a first strip of the solder is applied and exposed to the energysource, then a second strip is applied close to the first strip andexposed to the energy source and this process is repeated to provide aplurality of strip welds.
 8. The method of any one of claims 1 to 7wherein the biodegradable, biological solder is a protein solder.
 9. Themethod of claim 8 wherein the protein solder is a solid or a fluidsolder.
 10. The method according to any one of claims 1 to 9 wherein theenergy source is a laser.
 11. A method according to any one of claims 1to 10 wherein the solder incorporates a substance which absorbs theenergy from the energy source highly compared to the tissue.
 12. Amethod according to claim 11 wherein the substance is a dye.
 13. A fluidprotein solder composition comprising 100 to 120 mass % protein relativeto water, and a suitable solvent for the protein.
 14. A fluid proteinsolder composition according to claim 13 comprising 100 to 110 mass %protein relative to water, and a suitable solvent for the protein.
 15. Asubstantially solid protein solder comprising 120 to 230 mass % proteinrelative to water, and a suitable solvent for the protein.
 16. Asubstantially solid protein solder comprising 170 to 230 mass % proteinrelative to water, and a suitable solvent for the protein.
 17. Asubstantially solid protein solder comprising 210 mass % proteinrelative to water, and a suitable solvent for the protein.
 18. A kit forjoining tissues comprising, in a preferably sterile pack, a plurality ofstrips and/or shapes of a protein solder according to any one of claims15 to
 17. 19. A fluid protein solder composition comprising 100 to 120mass % protein relative to water, and a suitable solvent for theprotein, when used in a method according to any one of claims 1 to 10.20. A fluid protein solder composition according to claim 19 furthercomprising a substance which absorbs the energy from the energy sourcehighly compared to the tissue.
 21. A fluid protein solder compositionaccording to claim 20 wherein the substance is a dye.
 22. Asubstantially solid protein solder comprising 120 to 210 mass % proteinrelative to water, and a suitable solvent for the protein, when used ina method according to any one of claims 1 to
 10. 23. A substantiallysolid protein solder according to claim 22 further comprising asubstance which absorbs the energy from the energy source highlycompared to the tissue.
 24. A substantially solid protein solderaccording to claim 23 wherein the substance is a dye.